Compositions for topical treatment of microbial infections

ABSTRACT

The present invention provides compositions and methods for topical treatment of infections. The compositions comprise glycerol monolaurate or a derivative thereof, and are administered topically, for example, to treat viral, fungal or bacterial infections.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/866,722filed Apr. 19, 2013, which claims the benefit of U.S. provisionalapplication No. 61/636,203, filed Apr. 20, 2012, and U.S. provisionalapplication No. 61/650,755, filed May 23, 2012, each of which isincorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Some bacterial pathogens initiate human illnesses from intact or damagedmucosal or skin surfaces. Many of these pathogens are acquired fromother persons or animals, from endogenous sources, or from a myriad ofenvironmental sources. Once in humans, pathogens colonize surfacesprimarily as biofilms of organisms, defined as thin-films of organismsattached to host tissues, medical devices, and other bacteria throughcomplex networks of polysaccharides, proteins, and nucleic acids. Thesebacteria may also exist as planktonic (broth) cultures in some hosttissue environments, such as the bloodstream and mucosal secretions.Similarly, these potential pathogens may exist as either biofilms orplanktonic cultures in a myriad of non-living environments.

Glycerol monolaurate (GML) is a naturally occurring glycerol-basedcompound that has previously been shown to have anti-microbial,anti-viral, and anti-inflammatory properties. The present inventionprovides GML compositions and methods for the treatment of variousmicrobial infections and illnesses resulting from one or more microbialinfections.

SUMMARY OF THE INVENTION

Simple, inexpensive, and well tolerated methods and compositions areneeded for applying anti-microbial compounds such as GML at effectivelevels to skin and mucosal surfaces of humans and other vertebrates. Thepresent invention addresses this and other needs.

In one aspect, the present invention is directed to a compositioncomprising glycerol monolaurate (GML) or a derivative thereof, and avegetable oil. In one embodiment, the vegetable oil is palm, olive,corn, canola, coconut, soybean, or wheat, or a combination thereof. In afurther embodiment, the vegetable oil is present in the composition atabout 10% to about 99%, about 20% to about 90%, about 30% to about 80%,or about 40% to about 70%. In one embodiment, the composition comprisingGML or a derivative thereof and a vegetable oil further comprises apharmaceutically acceptable topical carrier, for example, petroleumjelly. In one embodiment, GML or a derivative thereof is present in thecomposition at a concentration from about 10 μg/mL to about 100 mg/mL,from about 50 μg/mL to about 50 mg/mL, from about 100 μg/mL to about 10mg/mL, or from about 500 μg/mL to about 5 mg/mL. In another embodiment,the composition comprising GML or a derivative thereof and a vegetableoil further comprises a cellulose derivative, for example eitherhydroxypropyl cellulose or hydroxyethyl cellulose, or a combinationthereof. In a further embodiment, the cellulose derivative is present inthe composition up to 1.25% w/w.

In another aspect, the present invention is directed to a compositioncomprising GML or a derivative thereof, and a non-aqueous gel. In oneembodiment, the composition comprising GML or a derivative thereof and anon-aqueous gel has a pH of about 4.0 to about 4.5. In one embodiment,the non-aqueous gel comprises polyethylene glycol, hydroxypropylcellulose, hydroxyethyl cellulose, or a combination thereof. In afurther embodiment, the polyethylene glycol is present at about 25% w/win the composition. In one embodiment, hydroxypropyl cellulose andhydroxyethyl cellulose are both present in the composition, each at aconcentration of about 1.25% w/w.

In one embodiment, the GML composition comprising a non-aqueous gelcomprises polyethylene glycol with a molecular weight range of about 300to about 4000. In a further embodiment, the polyethylene glycol has amolecular weight of about 400 or about 1000.

In one embodiment, the GML composition comprising a non-aqueous gelfurther comprises a topical carrier, e.g., petroleum jelly. In a furtherembodiment, the composition comprises a vegetable oil.

In one embodiment, the compositions described herein comprise GML or aderivative thereof at a concentration of about 0.001% (w/v) to about 10%(w/v) of the total composition. In a further embodiment, GML or aderivative thereof is present at about 0.005% (w/v) to about 5% (w/v) ofthe composition. In a further embodiment, GML or a derivative thereof ispresent at about 0.01 to about 1%. In a still further embodiment, GML ora derivative thereof is present at about 0.1% (w/v) to about 0.5% (w/v)of the composition.

In one embodiment, GML or a derivative thereof is present in thecomposition at a concentration of about 10 μg/mL to about 100 mg/mL. Ina further embodiment, GML or a derivative thereof comprises about 50μg/mL to about 50 mg/mL of the composition. In a further embodiment, GMLor a derivative thereof comprises about 100 μg/mL to about 10 mg/mL. Ina still further embodiment, GML or a derivative thereof comprises about500 μg/mL to about 5 mg/mL.

In one embodiment, the GML composition provided herein comprisespropylene glycol at a concentration of about 65% (w/w) to about 80%(w/w). In another embodiment, polyethylene glycol is present in thecomposition at a concentration of about 20% (w/w) to about 35% (w/w). Inone embodiment, both propylene glycol and polyethylene glycol arepresent in the topical composition.

In one embodiment, the composition comprises a cellulose derivative. Ina further embodiment, the composition comprises hydroxypropyl celluloseor hydroxyethyl cellulose. In a yet further embodiment, the cellulose ispresent at a concentration of about 0.1% (w/w) to about 5.0% (w/w).

In one embodiment, the GML composition comprises an aqueous solvent. Ina further embodiment, the aqueous solvent is water, saline, media, or acombination thereof.

In one embodiment, the pharmaceutically acceptable topical carrier ispetroleum jelly.

In one embodiment, the pH of the GML composition provided herein is fromabout 4.0 to about 5.5.

In some embodiments, the composition provided herein comprises one ormore accelerants. In a further embodiment, the accelerant is an organicacid, a chelator, an anti-bacterial agent, an anti-fungal agent, ananti-viral agent, or a combination thereof. In a further embodiment, theaccelerant is a chelator. In even a further embodiment, the accelerantis EDTA.

In another aspect, the GML composition provided herein hasanti-microbial, anti-viral, and/or anti-inflammatory activity. Forexample, in one embodiment, the composition provided herein is appliedtopically to humans and other vertebrates, for example for treatment ofa bacterial, fungal, or viral infection such as Gardnerella vaginalis orCandida albicans.

Accordingly, in one embodiment, the present invention provides methodsfor treating a microbial infection in a subject in need thereof. In oneembodiment, the method comprises topically administering to the subjectin need thereof, an effective amount of a GML composition providedherein. In one embodiment, the composition comprises GML or a derivativethereof, a vegetable oil, and a pharmaceutically acceptable topicalcarrier. In another embodiment, the composition comprises GML or aderivative thereof, a non-aqueous gel, and a pharmaceutically acceptabletopical carrier. In a further embodiment, the composition comprises GMLor a derivative thereof, a vegetable oil, a non-aqueous gel, and apharmaceutically acceptable topical carrier.

In one embodiment, the compositions disclosed herein are appliedtopically with the use of a sponge, wipe, or swab.

In one embodiment, the subject has a bacterial infection. In a furtherembodiment, the bacterial infection is Staphylococcus (such asStaphylococcus aureus); Streptococcus (such as Streptococcus pneumoniaeor Streptococcus agalactiae); Escherichia (such as Escherichia coli);Gardnerella (such as Gardnerella vaginalis); Clostridium (such asClostridium peifringens); Mycobacterium (such as Mycobacteriumtuberculosis or Mycobacterium phlei); or Chlamydia (such as Chlamydiatrachomatis).

In another embodiment, the subject treated with one of the GMLcompositions provided herein has a fungal infection. In a furtherembodiment, the fungal infection is Candida (such as Candida albicans),Microsporum species, Trichophyton species, Penicillium species, orAspergillus species.

In another embodiment, the method of the invention involvesadministering a second active agent selected from the group consistingof anti-fungal agents, anti-viral agents, and antibiotics.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of various concentrations of GML inolive oil on the growth of several microorganisms (measured as CFU/mL).Bars represent the following microorganisms, from left: Staphylococcusaureus MNPE (methicillin sensitive strain), S. aureus MW2 (methicillinresistant strain), Candida albicans, Streptococcus agalactiae, andGardnerella vaginalis.

FIG. 2 is a graph showing the effect of various concentrations of GML inpalm oil on the growth of several microorganisms (measured as CFU/mL).Bars represent the following microorganisms, from left: Staphylococcusaureus MNPE (methicillin sensitive strain), S. aureus MW2 (methicillinresistant strain), Candida albicans, Streptococcus agalactiae, andGardnerella vaginalis.

FIG. 3 is a graph showing the effect of various concentrations of GML incorn oil on the growth of several microorganisms (measured as CFU/mL).Bars represent the following microorganisms, from left: Staphylococcusaureus MNPE (methicillin sensitive strain), S. aureus MW2 (methicillinresistant strain), Candida albicans, Streptococcus agalactiae, andGardnerella vaginalis.

FIG. 4 is a set of scanning electron micrographs showing S. aureusgrowing as a biofilm on tampon fibers in cellulose acetate dialysistubing at 6000× magnification (left) or 9000× magnification (right).

FIG. 5 is a graph showing the accumulation of the superantigen TSST-1 inbiofilms grown on tampon fibers in cellulose acetate dialysis tubing.

FIG. 6 is a series of graphs showing the measured CFU/mL from biofilmsformed from S. aureus strains MN8 (methicillin sensitive strain, toppanels), MNWH (methicillin resistant strain, middle panels), and MW2(methicillin resistant strain, bottom panels) cultured in 96 wellplastic microtiter plates, in the presence or absence of the indicatedconcentrations of GML for 24 or 48 hours.

FIG. 7 is a series of graphs showing the measured biofilm absorbance at595 nm after crystal violet staining of biofilms, formed from S. aureusstrains MN8 (methicillin sensitive strain, top panels), MNWH(methicillin resistant strain, middle panels), and MW2 (methicillinresistant strain, bottom panels) cultured in 96 well plastic microtiterplates, for 24 or 48 hours, in the presence or absence of the indicatedconcentration of GML.

FIG. 8 is a set of graphs showing the measured CFU/mL (top) andabsorbance at 595 nm after crystal violet staining (bottom) fromHaemophilus influenzae biofilms cultured in 96 well plastic microtiterplates, for 24 or 48 hours, in the presence or absence of the indicatedconcentration of GML.

FIG. 9 is a set of graphs showing the measured CFU/mL (left) andabsorbance at 595 nm after crystal violet staining (right) from S.aureus or H. influenzae biofilms treated with 500 g/mL GML.

FIG. 10 is a graph showing CFU/mL from E. coli cultures grown for 24hours in the presence or absence of 100 μg/mL GML, and in the presenceof increasing concentrations of EDTA.

FIG. 11 is a graph showing CFU/mL from E. coli cultures grown for 24hours in 100 μg/mL GML alone, increasing concentrations of EDTA alone,or increasing concentrations of EDTA in the presence of 100 μg/mL GML.

FIG. 12 is a graph showing CFU/mL from S. aureus cultures grown at theindicated pH and at the indicated concentrations of GML.

FIG. 13 is a set of graphs showing CFU/mL from H. influenzae culturesgrown at the indicated pH, in the presence of the 1 μg/mL GML.

FIG. 14 is a graph showing CFU/mL from Pseudomonas aeruginosa culturesgrown at the indicated pH and at the indicated concentrations of GML.

FIG. 15 is a graph showing the effect of the indicated concentrations ofGML alone (black), GML in a 10% non-aqueous gel carrier (white) or a 25%non-aqueous gel carrier (grey), on the growth of S. aureus (CFU/mL).

FIG. 16 is a graph depicting the solubility of tenofovir (10 mg/mL) inGML at a pH ranging from 4.0 to 4.5. The absence of a bar indicates thatthe 10 mg/mL tenofovir was not soluble in the composition, while thepresence of a bar indicates that 10 mg/mL tenofovir was soluble in thecomposition

FIG. 17 is a set of graphs showing the bactericidal activity of theindicated forms of GML against S. pyogenes.

FIG. 18 is a graph showing the bactericidal activity (CFU/mL) of GMLcompared to lauric acid, in the presence of S. pyogenes.

FIG. 19 is a graph showing the bactericidal activity (CFU/mL) of GMLcompared to lauric acid, in the presence of S. aureus.

FIG. 20 is a graph showing superantigen production from S. aureus(TSST-1) or S. pyogenes (SPE A) in the presence of GML or lauric acid.

FIG. 21 is a graph showing the bactericidal activity of GML afterpre-treatment with S. aureus or S. pyogenes.

FIG. 22 is a graph showing the staphylococcal counts (CFU/mL) inanterior nares of three human subjects treated with 5% GML in anon-aqueous gel.

FIG. 23 is a graph showing the ability of 5% GML in a non-aqueous gel toreduce aerobic bacteria on human teeth and gum lines (CFU/mL).

FIG. 24 is a graph showing the presence of S. aureus on surgicalincision sites of rabbits 24 hours after swabbing with S. aureus MN8 andeither PBS or 5% GML

FIG. 25 shows the inflammation present at surgical incision sites of NewZealand white rabbits treated with S. aureus MN8 and then GML gel (left)or PBS (right) for 24 hours. Inflammation is apparent as dark greycoloring of the surgical site, as indicated by the arrows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides topical GML compositions and methods oftreatment with the compositions, e.g., by topical administration. Thecompositions and methods provided herein, in one embodiment, are usedfor treating infections topically, for example, by facilitating deliveryof effective amounts of GML or a derivative thereof to a skin or mucosalsurface of a subject, e.g., a human. Without wishing to be bound bytheory, it is believed that the compositions of the invention result ingreater patient compliance for topical self-administration due to theless irritating nature of the composition, relative to previouslyemployed topical formulations of anti-microbial and anti-viralcompounds.

As used herein, the term “antimicrobial” means effective in preventing,inhibiting, or arresting the growth or pathogenic effects of amicroorganism. “Microorganism” is used herein to mean any bacteria,virus, or fungus. In one embodiment, the formulations of the inventionare used to prevent, inhibit, or arrest the growth of one or more of thefollowing microorganisms: Staphylococcus aureus, Streptococcus (e.g., S.pyogenes, S. agalacticae or S, or S. pneumoniae) Haemophilus influenzae,Pseudomonas aeruginosa, Gardnerella vaginalis, Enterobacteriacae (e.g.,Escherichia coli), Clostridium peifringens, Chlamydia trachomatis,Candida albicans, Human Immunodeficiency Virus (HIV), or Herpes SimplexVirus (HSV).

“Anti-bacterial” or “anti-fungal,” as used herein, refer to inhibitionor arrest of the growth of a bacterium or fungus, a reduction in theseverity of or likelihood of developing a bacterial or fungal disease,inducing death of the bacterium or fungus or reduction or inhibition ofthe pathogenic effects of the respective bacterium or fungus.“Bactericidal” is used interchangeably with “anti-bacterial.”

“Anti-viral,” as used herein, refers to inhibition of viral infection orvirus replication, a reduction in the likelihood that a subject exposedto a virus will contract the viral disease, or a reduction in theseverity of the viral disease.

The term “effective amount,” as used herein, refers to an amount that issufficient to effect a beneficial or desired antimicrobial activity,including, without limitation, killing the microorganism or inhibitingmicrobial infection, growth or toxicity. An effective amount of GML isabout 10 μg/mL, about 100 μg/mL, about 1 mg/mL, about 10 mg/mL, about 50mg/mL, or about 100 mg/mL.

The terms “treat,” “treatment,” and “treating” refer to an approach forobtaining beneficial or desired results, for example, clinical results.For the purposes of this invention, beneficial or desired results mayinclude inhibiting or suppressing the growth of a microorganism orkilling a microorganism; inhibiting one or more processes through whicha microorganism infects a cell or subject; inhibiting or amelioratingthe disease or condition caused by a microbial infection; or acombination thereof. The terms “treat,” “treatment,” or “treating” alsorefer to prophylaxis of infection. In some embodiments, the formulationsof the invention are used to treat urinary tract infections, vaginalmicrobial infections, infections of the oral cavities such as thosecausing gum disease, post-surgical infections including respiratorytract infections, wound or surgical incision site infections, orinfections characterized by the production of toxins, including ToxicShock Syndrome.

“Prophylaxis,” as used herein, can mean complete prevention of aninfection or disease, or prevention of the development of symptoms ofthat infection or disease; a delay in the onset of an infection ordisease or its symptoms; or a decrease in the severity of a subsequentlydeveloped infection or disease or its symptoms.

As used herein, the term “subject” includes humans and other animals.The subject, in one embodiment, is a human.

“Topical,” as used herein, refers to the application of the compositionto any skin or mucosal surface. “Skin surface” refers to the protectiveouter covering of the body of a vertebrate, generally comprising a layerof epidermal cells and a layer of dermal cells. A “mucosal surface,” asused herein, refers to a tissue lining of an organ or body cavity thatsecretes mucous, including but not limited to oral, vaginal, rectal,gastrointestinal, and nasal surfaces. In one embodiment, theformulations of the invention are administered topically to the teethand gum, skin, nasal, or vaginal areas.

The term “pharmaceutically acceptable topical carrier,” as used herein,refers to a material, diluent, or vehicle that can be applied to skin ormucosal surfaces without undue toxicity, irritation, or allergicreaction.

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the present application includes both one and more than one suchexcipient.

As used herein, the term “vegetable oil” means a substance extractedfrom a plant or seed that exists in liquid form at room temperature.Suitable vegetable oils include, without limitation, palm, olive, corn,canola, coconut, soybean, wheat germ, jojoba, sunflower, sesame, peanut,cottonseed, safflower, soybean, rapeseed, almond, beech nut, cashew,hazelnut, macadamia, mongongo nut, pecan, pine nut, pistachio, walnut,grapefruit seed, lemon, orange, bitter gourd, bottle gourd, buffalogourd, butternut squash seed, egusi seed, pumpkin seed, watermelon seed,acai, black seed, blackcurrant seed, borange seed, evening primrose,flaxseed, eucalyptus, amaranth, apricot, apple seed, argan, avocado,babassu, coriander seed, grape seed, mustard, poppyseed, rice bran,castor, or mixtures thereof. Mixtures can be, by way of example andwithout limitation, a combination of olive oil and soybean oil, acombination of coconut oil and wheat germ oil, or a combination ofjojoba oil, palm oil, and castor oil. Mixtures of vegetable oils can bebinary, ternary, quaternary, or higher mixtures.

The term “accelerant,” as used herein, refers to a compound, substance,liquid, powder, or mixture that, when added to the composition, has theeffect of enhancing or contributing to the antimicrobial properties ofthe composition. Accelerants may be an organic acid including, withoutlimitation, lactic acid, ascorbic acid, citric acid, formic acid,benzoic acid, and oxalic acid. The accelerant, in another embodiment, isa chelator, and in one embodiment, is selected fromethylenediaminetetraacetic acid (EDTA), dimercaprol, dimercaptosuccinicacid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), alpha lipoicacid (ALA), or combinations thereof. In another embodiment, theaccelerant is selected from an antibiotic agent, anti-fungal agent,anti-viral agent, or combination thereof. Antibiotics for use with theinvention, for example, include aminoglycosides, carbacephems,cephalosporins, glycopeptides, lincosamides, lipopetides, macrolides,monobactams, nitrofurans, penicillins, polypetides, quinolones,sulfuramides, and tetracyclines. Anti-fungal agents include, withoutlimitation, those of the azole class, polyene class, or echinocaninsclass, nucleoside analogues, allylamines, griseofulvin, tolnaftate, orselenium compounds. Anti-viral agents include, for example and withoutlimitation, acyclovir, ganciclovir, valganciclovir, abacavir, enofovir,lamivudine, emtricitabine, zidovudine, tenofovir, efavirenz,raltegravir, enfuvirdide, maraviroc, ribavirin, amantadine, rimantadine,interferon, oseltamivir, and zanamivir.

As used herein, the term “cellulose derivative” refers to any acellulose-based compound and may include, for example, hydroxyethylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose,hydroxypropyl methyl cellulose, or cellulose acetate.

The term “biofilm,” as used herein, means an aggregate ofmicroorganisms, usually bacterial, adhered to one another and growing ona surface. The microbial cells in the biofilm typically produce anextracellular matrix known as an extracellular polymeric substance.Often, this matrix and the density of the aggregate itself significantlyincrease the antibiotic resistance of the bacteria in the biofilm.Biofilms can be involved in UTis, ear infections, and dental diseasessuch as gingivitis, and can also form on the surface of implanteddevices including prostheses, catheters, or heart valves.

In one aspect, the present invention provides a topical compositioncomprising glycerol monolaurate (GML) or a derivative thereof. In afurther embodiment, the composition comprises a vegetable oil or anon-aqueous gel, or a combination thereof. The non-aqueous gel, in oneembodiment, comprises a cellulose derivative. The topical compositionprovided herein, in one embodiment, comprises a pharmaceuticallyacceptable topical carrier.

In one embodiment, the composition provided herein comprises themonoglyceride GML. GML is a fatty acid ester of glycerol, derivative oflauric acid, with the chemical formula C15H3004. GML is also known inthe art as glyceryl laurate or monolaurin. GML is found naturally inbreast milk and some plants, and is used as a food and cosmeticadditive. GML and other glycerides are listed in the GenerallyRecognized as Safe Substances database by the US Food and DrugAdministration. GML and related compounds have been previously disclosedin U.S. patent application Ser. No. 10/579,108 (filed Nov. 10, 2004) andSer. No. 11/195,239 (filed Aug. 2, 2005), the disclosures of each ofwhich are herein incorporated by reference for all purposes.

GML can be synthesized in multiple forms including both R and S opticalisomers, as well as forms with lauric acid in the 1/3-position and inthe 2-position. The composition provided herein, in one embodiment,comprises the R isomer of GML. In another embodiment, the compositionprovided herein comprises the S isomer of GML. In yet anotherembodiment, a racemic mixture of isomers is provided in the composition.

Similarly, the topical composition may comprise GML with lauric acid atthe 1/3 position, GML with lauric acid at the 2-position, or acombination thereof. R and S isomers of each form, and racemic mixturesthereof, are amenable for use with the present invention.

The chemical structure of GML with lauric acid in the 1/3-position is

Glycerol monolaurate (GML) 1/3-position

The chemical structure of GML with lauric acid in the 2-position is:

Glycerol monolaurate (GML) 2-position

In another embodiment, the topical composition comprises a GMLderivative, for example a compound selected from one of Formulae I-VI.Examples of such compounds include, by way of example and withoutlimitation, glycerol monocaprylate, glycerol monocaprate, glycerolmonomyristate, glycerol monopalmitate, and dodecyl glycerol.

wherein each occurrence of X is independently —O— or —S—; and n is aninteger from 5 to 20 (inclusive).

In another embodiment, the topical composition comprises at least onederivative of GML, and the at least one derivative is a compound ofeither Formula V or Formula VI. Examples of such compounds include, butare not limited to, glycerol dilaurate, glycerol dicaprylate, glyceroldimyristate, glycerol trilaurate, and glycerol tripalmitate.

wherein each occurrence of X is independently —O— or —S—; andeach occurrence of n is independently an integer from 5 to 20(inclusive).

In one embodiment, a compound of Formula I, II, III or IV is present inthe topical composition of the invention, and at least one —X— is —S—.In one embodiment, one occurrence of —X— is —S— and the remainingoccurrences of —X— are —O—.

In one embodiment, a compound of Formula V or VI is present in thetopical composition of the invention, each occurrence of n is 10, and atleast one —X— is —O—.

The topical composition provided herein, in one embodiment, comprisesGML and a GML derivative. For example, in one embodiment, the topicalcomposition provided herein comprises GML and a compound of Formula VI.In a further embodiment, each occurrence of n is 10 and at least one —X—is —O—.

In one embodiment, the topical composition comprises GML or a derivativethereof from about 0.001% (w/v) to about 10% (w/v) of the composition.In a further embodiment, GML or a derivative thereof comprises about0.005% (w/v) to about 5% (w/v) of the composition. In a still furtherembodiment, GML or a derivative thereof comprises about 0.01%/(w/v) toabout 1.0% (w/v) of the composition. In yet a further embodiment, GML ora derivative thereof comprises about 0.05% (w/v) to about 0.5% (w/v) ofthe composition.

In another embodiment, the topical composition comprises GML orderivative thereof at a concentration of about 10 μg/mL to about 100mg/mL. In a further embodiment, the topical composition comprises GML orderivative thereof at a concentration of about 50 μg/mL to about 50mg/mL. In a further embodiment, the topical composition comprises GML orderivative thereof at a concentration of about 100 μg/mL to about 10mg/mL. In yet a further embodiment, the topical composition comprisesGML or a derivative thereof at a concentration of about 500 μg/mL toabout 5 mg/mL.

In one embodiment, the topical composition comprises GML or derivativethereof at a concentration of about 10 μg/mL, about 50 μg/mL, about 100μg/mL, about 500 μg/mL, about 1 mg/mL, about 5 mg/mL, about 10 mg/mL,about 50 mg/mL, or about 100 mg/mL.

The amount of GML or derivative thereof in the composition can betailored accordingly to the indication/disease being treated as well asthe characteristics of the subject being treated. The amount of GML inthe composition may vary depending on, for example, the nature of theinfection or illness; the site of administration; the subject's medicalhistory, subject weight, age, sex, and surface area being treated; andwhether the subject is receiving any other medications.

As provided above, in one aspect, the present invention is directed to atopical composition comprising GML or a derivative thereof. In oneembodiment, the topical composition comprises at least one glycol. Forexample, in one embodiment, the topical composition comprises propyleneglycol, polyethylene glycol, or a combination thereof. In oneembodiment, the polyethylene glycol has a molecular weight (MW) rangefrom about 300 to about 10,000. In a further embodiment, thepolyethylene glycol has a molecular weight of about 300 to about 1,000.In a still further embodiment, the polyethylene glycol has a molecularweight of about 400.

In one embodiment, polyethylene glycol is present in the topicalcomposition. In a further embodiment, the polyethylene glycol has a MWof about 400, about 500 or about 1,000. In one embodiment, thepolyethylene glycol is present in the topical composition at aconcentration (w/w) of about 15% to about 50%, about 20% to about 40%,or about 25% to about 35%, for example, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, or about 50%. In a furtherembodiment, both propylene glycol and polyethylene glycol are present inthe topical composition. In a further embodiment, propylene glycol ispresent at a concentration of about 70% to about 80% and polyethyleneglycol is present at a concentration of about 20% to about 30%. In evena further embodiment, the polyethylene glycol is polyethylene glycol400.

In another embodiment, a topical composition comprising GML or aderivative thereof is provided. In a further embodiment, propyleneglycol is present in the composition. In yet a further embodiment,propylene glycol is present in the composition at a concentration ofabout 60% to about 80%/a, for example, about 60%, about 65%, about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, or about 80%.

In another embodiment, a topical composition comprising GML or aderivative thereof is provided. In one embodiment, the topicalcomposition comprises at least one cellulose derivative. In a furtherembodiment, the composition comprises one cellulose derivative or twocellulose derivatives. In one embodiment, the cellulose derivative ishydroxypropyl cellulose. In another embodiment, the cellulose derivativeis hydroxyethyl cellulose, carboxymethyl cellulose or hydroxymethylcellulose. In yet another embodiment, the composition comprise acombination of hydroxyethyl cellulose and hydroxypropyl cellulose. Inone embodiment, the cellulose derivative is present at a concentrationof about 0.1% (w/w) to about 5.0% (w/w). In a further embodiment,multiple cellulose derivatives are present in the composition at thesame concentration. In a further embodiment, two cellulose derivativesare present, and each is present at a concentration of about 1.25%(w/w). Cellulose derivatives include, for example, hydroxyethylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose,hydroxypropyl methyl cellulose, or cellulose acetate.

In one embodiment, the topical composition provided herein comprises GMLor a derivative thereof, at least one cellulose derivative, propyleneglycol and polyethylene glycol.

In another embodiment, a topical composition comprising GML or aderivative thereof is provided. In a further embodiment, the compositioncomprises at least one vegetable oil, for example, at least one of thevegetable oils described above (e.g., palm oil, olive oil, corn oil). Inone embodiment, the vegetable oil is present in the composition at aconcentration of about 0.1% (w/w) to about 10% (w/w). In a furtherembodiment, the vegetable oil is present in the composition at aconcentration of about 1% (w/w) to about 8% (w/w). In a furtherembodiment, the vegetable oil is present in the composition at aconcentration of about 1% (w/w) to about 6% (w/w). In a furtherembodiment, the vegetable oil is present in the composition at aconcentration of about 1% (w/w) to about 4% (w/w). In one embodiment,the vegetable oil is present in the composition at a concentration ofabout 0.1% (w/w), about 0.5% (w/w) about 1.0%0/(w/w), about 1.25% (w/w),about 1.5% (w/w), about 1.75% (w/w), or about 2.0% (w/w).

In one embodiment, the topical composition provided herein comprises avegetable oil and at least one cellulose derivative. For example, in oneembodiment, the topical composition comprises hydroxypropyl celluloseand a vegetable oil, or hydroxyethyl cellulose and a vegetable oil, or acombination of hydroxypropyl cellulose, hydroxyethyl cellulose, and avegetable oil. In one embodiment, the cellulose derivative and thevegetable oil (e.g., palm, oil or corn oil), are each present at thesame concentration (w/w). In a further embodiment, the cellulosederivative and the vegetable oil are each present in the composition atabout 1% (w/w) to about 5% (w/w). In even a further embodiment, thecellulose derivative is a combination of hydroxypropyl cellulose andhydroxyethyl cellulose, and each is present in the composition at about1.25% (w/w). In one embodiment, the composition comprises a vegetableoil and two cellulose derivatives. In a further embodiment, the twocellulose derivatives are hydroxypropyl cellulose and hydroxyethylcellulose, and the total concentration of cellulose derivatives in thecomposition is about 1.25% (w/w). Cellulose derivatives include, forexample, hydroxyethyl cellulose, hydroxypropyl cellulose,methylcellulose, ethylcellulose, hydroxypropyl methyl cellulose, orcellulose acetate.

In some embodiments, the topical composition provided herein comprisesone or more accelerants. In a further embodiment, the accelerant is anorganic acid, a chelator, an anti-bacterial agent, an anti-fungal agent,an anti-viral agent, or a combination thereof. In a further embodiment,the accelerant is a chelator. In even a further embodiment, theaccelerant is EDTA.

The accelerant, in one embodiment, is EDTA. In a further embodiment, theGML composition provided herein comprises EDTA at a concentration ofabout 0.00005 M, about 0.0005 M, about 0.005 or about 0.05 M. In anotherembodiment, a chelator is present in the composition at a concentrationof about 0.00005 M to about 0.05 M, about 0.0005 M to about 0.005 M, orabout 0.005 to about 0.05 M.

In one embodiment, the topical composition comprises both a vegetableoil and an accelerant, for example palm oil and EDTA. In anotherembodiment, the accelerant is an organic acid and is present in theformulation with a vegetable oil. In one embodiment, the topicalcomposition provided herein comprises an accelerant and a non-aqueousgel, for example a gel comprising a cellulose derivative. In anotherembodiment, the topical composition comprises GML or a derivativethereof, a vegetable oil, a non-aqueous gel (e.g., a gel comprising oneor more cellulose derivatives) and an accelerant.

In one embodiment, the composition contains at least onepharmaceutically acceptable excipient. Pharmaceutically acceptableexcipients are well known to those skilled in the art and may includebuffers (e.g., phosphate buffer and citrate buffer), amino acids,alcohols, proteins such as serum albumin, parabens (e.g.,methylparaben), or mannitol.

In one embodiment, the pH of the composition is from about 3.5 to about7.0. In a further embodiment, the pH of the composition is from about4.0 to about 6.0. In a still further embodiment, the pH of thecomposition is from about 4.0 to about 4.5.

In one embodiment, the composition provided herein comprises GML or aderivative thereof and a pharmaceutically acceptable topical carrier. Inone embodiment, the pharmaceutically acceptable topical carrier is a mixof hydrocarbons such as, for example, paraffin wax or petroleum jelly.Petroleum jelly is any water-insoluble, hydrophobic, semi-solid mixtureof hydrocarbons. The pharmaceutically acceptable topical carrier can beadded to any of the formulations described herein.

In another embodiment, the composition comprises an aqueous solvent.Compositions comprising an aqueous solvent may or may not include apharmaceutically acceptable topical carrier. In one embodiment, theaqueous solvent is present, and is water, saline, growth medium (e.g.,microbial culture medium or cell culture medium), or a combinationthereof. In a further embodiment, both an aqueous solvent andpharmaceutically acceptable topical carrier are present in the topicalcomposition. In even a further embodiment, the topical compositioncomprises at least one cellulose derivative.

In one embodiment, the composition comprises bacterial culture mediasuch as Todd Hewitt media as the aqueous solvent. In one embodiment, theaqueous solvent is present at a concentration of about 1% (w/w) to about25% (w/w). In a further embodiment, the aqueous solvent is about 2%(w/w) to 5% (w/w) of the composition.

In one embodiment, the composition is a liquid solution. In anotherembodiment, the composition is a gel. In another embodiment, thecomposition is a solid, semi-solid, foam, wax, cream, or lotion.

In one embodiment, the composition comprises one of the formulationsprovided in Table 1. The vegetable oil, in one embodiment, is palm,olive or corn vegetable oil. It should be noted that Table 1 is merelyexemplary of the composition components and concentrations that can beused with the present invention.

TABLE 1 Exemplary GML Formulations Concentration Formulation Components(if applicable) 1 GML 0.001%-10% w/v (10 μg/mL-100 mg/mL) Cellulosederivative 0.1% (w/w) to 5.0% (w/w) Vegetable oil up to 100% w/v 2 GML0.001%-10% w/v (10 μg/mL-100 mg/mL) Non-aqueous Gel 10%-25% w/vPolyethylene glycol 400 (20% to 35% (w/w)) Propylene glycol (65% to 80%(w/w)) Cellulose derivative (0.5% to 5.0% (w/w)) Water or saline up to100% w/v 3 GML 0.001%-10% w/v (10 μg/mL-100 mg/mL) Non-aqueous Gel10%-25% w/v Polyethylene glycol 400 (20% to 35% (w/w)) Propylene glycol(65% to 80% (w/w)) Cellulose derivative (0.5% to 5.0% (w/w)) Water orsaline 1% to 25% w/v Vegetable oil up to 100% w/v 4 GML 0.001%-10% w/v(10 μg/mL-100 mg/mL) Non-aqueous Gel 10%-25% w/v Polyethylene glycol 400(20% to 35% (w/w)) Propylene glycol (65% to 80% (w/w)) Cellulosederivative (0.5% to 5.0% (w/w)) Vaseline up to 100% w/v 5 GML 5% w/vNon-aqueous Gel 10% w/v Polyethylene glycol 400 (25% (w/w)) Propyleneglycol (73.55% (w/w)) Cellulose derivative (1.25% (w/w)) Water 85% w/v 6GML 100 μg/mL Vegetable oil up to 100% w/v

In one aspect, the present invention provides a method of treating amicrobial infection in a subject in need thereof. The microbialinfection, in one embodiment, is a bacterial, viral, or fungalinfection, or a combination thereof.

Without wishing to be bound by theory, the GML topical compositionsdescribed herein are less irritating than currently approvedantimicrobial compositions, therefore resulting in a more favorablepatient compliance rate, as compared to other antimicrobial compositionspresently used in the art.

In one embodiment, the method comprises administering to the subject atopical composition comprising GML or a derivative thereof, as describedherein. In one embodiment, the method comprises topically administeringto the subject an effective amount of a composition comprising GML or aderivative thereof (e.g., a compound of one of Formulae I-VI), avegetable oil, and a pharmaceutically acceptable topical carrier. Inanother embodiment, the method comprises topically administering aneffective amount of a composition comprising GML, a non-aqueous gel, anda pharmaceutically acceptable topical carrier. In yet anotherembodiment, the method comprises administering to the subject one of thecompositions provided in Table 1.

In one embodiment, the method of treating a microbial infectioncomprises applying an effective amount of one or more of the GMLcompositions described herein to at least one skin or mucosal surface ofa subject.

In some embodiments, the composition is applied to or impregnated in awipe, sponge, swab, or other material, and then applied to the skin ormucosal surface of the subject using the respective material. As usedherein, the term “swab” refers to a material suitable for applying aliquid, gel, wax, cream, or lotion to a skin or mucosal surface, or theact of applying a liquid, gel, wax, cream, or lotion to the skin ormucosal surface, or the act of collecting a liquid, gel, wax, cream,lotion, or fluid from the skin or mucosal surface. In some embodiments,the material is attached to a holder, for example a stick, wire, rod, orapplicator. In further embodiments, the material attached to a holder isattached at one or both ends thereof. In some embodiments, the wipe,sponge, swab, or other material is pre-loaded or packaged together withthe composition.

Certain bacteria have been shown to be resistant to GML's antibacterialeffect. Such bacteria include those with a dense LPS layer e.g., speciesof Enterobacteriaceae for example, E. coli, as well as Pseudomonasaeruginosa. In addition, the antimicrobial activity of GML can beinhibited by the production of lipases or other hydrolyse enzymes suchas the esterase GEH, which is produced by S. aureus.

Without wishing to be bound by theory, it is thought that GML inhibitsmicrobial infection through one or more of several mechanisms thatinclude, but are not limited to, direct microbial toxicity; inhibitingentry of the infectious microorganism into the vertebrate cell;inhibiting growth of the microorganism; inhibiting production oractivity of virulence factors such as toxins; stabilizing the vertebratecells; or inhibiting induction of inflammatory or immunostimulatorymediators that otherwise enhance the infectious process.

Bacteria use two-component signal transduction systems to respond andadapt to environmental changes as well as produce virulence factors.Without wishing to be bound by theory, GML is believed to interfere withbacterial signal transduction, either directly or indirectly, throughinteraction with bacterial plasma membranes. In one embodiment, GML'sbactericidal effect is mediated at least in part by interactions at thebacterial plasma membrane. In a further embodiment, GML can be detectedin association with the bacterial plasma membrane, but cannot bedetected in association with the cytoplasm.

In one embodiment, direct GML-mediated interruption of bacterialmembranes includes interference with the localization of signalingproteins within the membrane, or interference with ligand binding tosignaling proteins. In one embodiment, GML has an indirect effect on atwo-component signal transduction system and the effect is selected frommodifications to membrane structure that interfere with the ability oftransmembrane proteins to perform signaling functions; dissipation ofthe bacterial plasma membrane potential; and alterations of pH gradientsacross the membranes.

In one embodiment, the indirect effect described above is mediatedthrough one or more tetramic acids, for example those produced by P.aeruginosa and certain lactobacillus strains. Tetramic acids made bythese organisms contain a 2,4 pyrrolidinedione ring and a 12 carbon sidechain. Their properties include broad spectrum antibacterial effects andanti-inflammatory activities. Without wishing to be bound by theory,mechanistic similarities between tetramic acids and GML may explain whyP. aeruginosa and lactobacilli are highly resistant to GML antimicrobialactivities. For P. aeruginosa, tetramic acids are important for thehomoserine lactone quorum sensing system. For example, P. aeruginosagrown in the presence of high concentrations of GML (>2000 μg/ml) at pH7.0 appears to have up-regulated production of numerous virulencefactors including pigments, consistent with effects associated withactivation of the quorum sensing system.

Exemplary two-component systems found in S. aureus include the agrregulatory system and WalK/R. It has been shown that GML affects the agrregulatory system, which regulates several virulence factors in S.aureus. WalK/R is essential for microbial viability. Without wishing tobe bound by theory, one or more two-component systems critical formicrobial viability such as WalK/R, for example, may be directlyinhibited by GML at higher doses, resulting in rapid death of themicrobes.

Similar to GML's putative effects on bacterial plasma membranes, GML hasbeen shown to inactivate certain viruses by disrupting viral lipidenvelopes [Thormar et al. (1994) Ann NY Acad Sci 724; 465].

In one embodiment, the methods described herein are used to treat apatient with a vaginal microbial infection. In a further embodiment, thevaginal microbial infection is vulvovaginal candidiasis (VVC) orbacterial vaginosis (BV). Women with BV are at risk for pelvicinflammatory disease, endometritis, and vaginal cuff cellulitis, andpregnant women with BV are at further risk of low birth weight, pre-termlabor, pre-term delivery, and chorioamnionitis. In patients with VVC orBV, the vaginal flora, which is normally dominated by Lactobacillusspecies, becomes altered such that other bacterial and/or fungal speciesdominate. Gardnerella vaginalis and other anaerobic bacteria arecommonly associated with BV; Candida species, usually C. albicans, areassociated with VVC. Accordingly, in one embodiment, the methodsprovided herein are used to treat a patient with an anaerobic bacterialinfection. In a further embodiment, the infection is a Gardnerellavaginalis or Candida infection (e.g., C. albicans).

The GML compositions provided herein, in one embodiment, are used inmethods to inhibit the production of toxins. For example, in oneembodiment, a method is provided to inhibit a bacterial toxin and/orreduce illness associated with a bacterial toxin. In a furtherembodiment, the method comprises applying one or more of the topicalcompositions described herein to a tampon or wound dressing, which issubsequently used by the subject, or applied to the subject. In afurther embodiment the bacterial illness treated by the methodsdescribed herein is Toxic Shock Syndrome (TSS), which is caused byproduction of TSS toxin 1 (TSST-1) or, more rarely, other toxins such asenterotoxin A, B, and C, by S aureus. The symptoms and sequelae of TSSmay include an acute fever, rash, hypotension, malaise, multiple organfailure, coma, desquamation of the skin, or death. Most cases of TSS areassociated with the use of tampons during menstruation, although TSS canoccur in any individual with a S. aureus infection, particularly anindividual with a skin wound.

Urinary tract infections (UTIs) are particularly common in women andelderly individuals. UTIs typically begin in the lower urinary tract(i.e., the urethra and bladder), and are generally treated withantibiotics after the onset of symptoms. If left untreated, a UTI canspread to the kidney and result in permanent kidney damage. UTIs aretypically caused by Escherichia coli, but can also be caused by otherEnterobacteriaceae, Staphylococcus aureus, other gram positive bacteriaor, more rarely, viruses or fungal species. In one embodiment, themethods described herein are used to treat a subject having a urinarytract infection. The method comprises, in one embodiment, topicallyapplying to a skin or mucosal surface of the patient, one or more of thecompositions described herein. In a further embodiment, the patient hasundergone long-term antibiotic therapy prior to the topical applicationof the composition.

In order to establish infection in a host subject, viruses such as HIVand SIV are believed to require an initial inflammatory response thatresults in recruitment of CD4+ T cells which are subsequently infectedby the virus, to the site of infection. In one embodiment, the methodsdescribed herein are used to treat HIV and/or SIV infections. The methodcomprises, in one embodiment, topically applying to a skin or mucosalsurface of the patient, one or more of the compositions describedherein. In a further embodiment, the composition is administeredintra-vaginally.

It is estimated that there are more than 500,000 cases of post-surgicalS. aureus infections yearly in the United States, and it has been shownthat 80% of such infections result from the same bacterium that is foundin patients' anterior nares. Additionally, there have been significantoutbreaks of streptococcal pharyngitis and streptococcal toxic shocksyndrome associated with upper respiratory tract infection withStreptococcus pyogenes, for example an outbreak of the M3 strain. Thesefacts suggest that nasal decolonization, when combined with possibledecolonization of other parts of the upper respiratory tract and use ofsurgical scrubs that kill pathogens on surgical sites, may be effectivein preventing and treating post-surgical infections and otherrespiratory tract infections. In some embodiments, the GML compositionsprovided herein are used in methods to decolonize the respiratory tract,other mucosal surfaces, or surgical incision sites in order to reducestreptococcal pharyngitis, streptococcal toxic shock syndrome orpost-surgical S. aureus infections. In some embodiments, the methodcomprises applying one or more of the compositions provided herein tothe anterior nares of a subject. For example, in one embodiment, 1 mg/mLGML in a 10% non-aqueous gel is applied to a swab and the swab isrotated around each nare up to the nasal bone 3 times.

It has been reported that oral streptococci are implicated in gumdisease and dental caries, and, in susceptible individuals, infectiveendocarditis. The GML compositions provided herein are used in someembodiments to prevent or treat streptococcal infections that lead todental caries, gum disease, and infective endocarditis. The method inone embodiment comprises applying to the teeth and gum lines of asubject one or more of the compositions provided herein. For example, inone embodiment, 1 mg/mL GML in a 5% non-aqueous gel is applied to theteeth and gum lines of a subject using a swab.

In some embodiments, the subject has a bacterial infection. Bacterialinfections that are treatable with the topical compositions providedherein include, but are not limited to, infections caused by thefollowing bacteria: Staphylococci (e.g., S. aureus, S. intermedius, S.epidermidis), Group A Streptococcus (e.g., S. pyogenes), Group BStreptococcus (e.g., S. agalacticae), Groups C, F, and G Streptococcus,Streptococcus pneumoniae, Bacillus anthracis, Peptostreptococcusspecies, Clostridium peifringes, Neisseriae gonorrheae, Chlamydiatrachomatis, Haemophilus influenzae, Pseudomonas aeruginosa,Helicobacter pylori, Gardnerella vaginalis, Bacteroides fragilis,Burkholderia cepacia, Bordatella bronchiseptica, Campylobacter jejuni,Enterobacteriacae (e.g., Escherichia coli) Pasteurella multocida, andMycobacterium (e.g., M. tuberculosis and M phlei).

Additionally, the topical compositions described herein, in oneembodiment, are used to treat one or more bacterial infections caused byone or more of the bacteria listed in Table 2. Table 2 shows the resultsof experiments testing the anti-bacterial activity of GML againstvarious bacteria grown under optimal growth conditions. Burkholderiacenocepacia, which used to be named Pseudomonas cepacia and is relatedto Pseudomonas aeruginosa, was killed by GML at concentrations of 500μg/mL. Mycobacterial species typically produce large amounts of complexfatty acids. However, these organisms were killed by GML atconcentrations of ≧50 μg/mL. In addition to inhibiting the growthgram-positive bacteria, GML inhibited exotoxin production independentlyfrom inhibition of growth for all such organisms tested (Staphylococcusaureus, Streptococcus pyogenes, Streptococcus agalactiae, groups C, F,and G streptococci, and Clostridium perfringens). The most susceptibleorganisms to killing by GML were Peptostreptococcus species, Clostridiumperfringens, Bordetella bronchiseptica, and Campylobacter jejuni, all ofwhich were killed by GML (1 μg/mL).

TABLE 2 Spectrum of antibacterial activity of GML Average BactericidalGram or Concentration Other Oxygen Strains of GML Bacterium StainTolerance Tested (μg/ml) Staphylococcus Positive Aerobe 54 300 aureusStreptococcus Positive Aerotolerant 4 30 pyogenes Anaerobe StreptococcusPositive Aerotolerant 3 30 agalactiae Anaerobe Group C PositiveAerotolerant 1 30 Streptococcus Anaerobe Group F Positive Aerotolerant 120 Streptococcus Anaerobe Group G Positive Aerotolerant 1 50Streptococcus Anaerobe Streptococcus suis Positive Aerotolerant 1 50Anaerobe Streptococcus Positive Aerotolerant 1 50 sanguinis AnaerobeStreptococcus Positive Aerotolerant 2 10 pneumoniae Anaerobe SerotypeIII Enterococcus Positive Aerotolerant 1 100 faecalis Anaerobe ListeriaPositive Aerobe 1 50 monocytogenes Bacillus anthracis Positive Aerobe 150 Sterne Bacillus cereus Positive Aerobe 1 50 PeptostreptococcusPositive Anaerobe 1 1 species Clostridium Positive Anaerobe 1 1perfringens Neisseria Negative Aerobe 1 20 gonorrhoeae HaemophilusNegative Aerobe 2 50 influenzae Non-typable Gardnerella Negative Aerobe2 10 vaginalis Campylobacter Negative Aerobe 1 1 jejuni BordetellaNegative Aerobe 1 1 bronchiseptica Pseudomonas Negative Aerobe 1 NotSusceptible aeruginosa Burkholderia Negative Aerobe 1 500 cenocepaciaPasteurella Negative Aerobe 1 500 multocida Prevotella Negative Anaerobe1 50 melaninogenica Bacteroides fragilis Negative Anaerobe 2 50Fusobacterium Negative Anaerobe 1 50 species Escherichia coli NegativeAerobe 2 Not Susceptible Salmonella Negative Aerobe 1 Not Susceptibleminnesota Enterobacter Negative Aerobe 1 Not Susceptible aerogenesProteus vulgaris Negative Aerobe 1 Not Susceptible Shigella sonniNegative Aerobe 1 Not Susceptible Klebsiella Negative Aerobe 1 NotSusceptible pneunoniae Mycobacterium Acid Fast Aerobe 1 100 phleiMycobacterium Acid Fast Aerobe 1 100 tuberculosis Mycoplasma Cell WallAerobe 1 1 hominis deficient

In some embodiments, the subject to be treated with one or more of thetopical compositions provided herein has a viral infection. In a furtherembodiment, the viral infection is caused by one or more of thefollowing viruses, or class of viruses: influenza virus, herpesviruses(e.g., Herpes Simplex Virus 2), lentiviruses (e.g., HumanImmunodeficiency virus).

In some embodiments, the subject to be treated with one or more of thetopical compositions provided herein has a fungal infection. In afurther embodiment, the fungal infection is caused by one or more of thefollowing organism species: Candida species (e.g. C. albicans),Microsporum species, Trichophyton species, Epidermophyton floccosum,Penicillium species, Aspergillus species, Trichomonas vaginalis.

Methods of identifying and diagnosing a bacterial, viral, or fungalinfection are generally known by those skilled in the art. To assesswhether the formulations disclosed herein are useful to treat aninfection, methods known to those of ordinary skill in the art may beemployed. For example, a BV infection prior to, and after treatment, maybe assessed by microscopic examination of vaginal cells.

In one embodiment, a method is provided to remove or kill a biofilmcomprising one or more microorganisms. Biofilms can be involved in UTIs,ear infections, and dental diseases such as gingivitis, and can alsoform on the surface of implanted devices including prostheses,catheters, or heart valves. In one embodiment, the method comprisesadministering the topical composition by applying it directly to thebiofilm.

In some embodiments, the methods of the invention comprise administeringa second active agent, along with GML or a derivative of GML. Theadditional active agent may be present in the compositions describedherein, or may be administered separately. In one embodiment, the one ormore additional active agents prior to, or after, the topical GMLcomposition is administered. For example, the two active agents may betopically administered serially, or administered serially by differentroutes of administration.

In one embodiment, the additional active agent(s) is administeredbefore, during, or after administration of the composition of theinvention. In another embodiment, the additional active agent(s) isadministered by the same route as the composition or by a differentroute. For example, the additional active agent(s), in one embodiment,is administered by one of the following routes of administration:topical, intranasal, intradermal, intravenous, intramuscular, oral,vaginal, rectal, otic, ophthalmic, subcutaneous. The dose of additionalactive agents depends on, for example, the nature of the infection orillness; the site of administration; subject weight, age, sex, andsurface area; concomitant medications; and medical judgment.

Additional active agents include, for example, antibiotics, anti-viralagents, and anti-fungal agents. Antibiotics include, without limitation,aminoglycosides, carbacephems, cephalosporins, glycopeptides,lincosamides, lipopetides, macrolides, monobactams, nitrofurans,penicillins, polypetides, quinolones, sulfuramides, and tetracyclines.Anti-fungal agents include, without limitation, those of the azoleclass, polyene class, or echinocanins class, nucleoside analogues,allylamines, griseofulvin, tolnaftate, or selenium compounds. Anti-viralagents include, for example and without limitation, acyclovir,ganciclovir, valganciclovir, abacavir, enofovir, lamivudine,emtricitabine, zidovudine, tenofovir, efavirenz, raltegravir,enfuvirdide, maraviroc, ribavirin, amantadine, rimantadine, interferon,oseltamivir, and zanamivir.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1 Antimicrobial Effects of GML in Vegetable Oil

A study was undertaken to assess the ability of various concentrationsof GML in olive, palm, or corn oil to inhibit the growth of severalbacterial or fungal microorganisms in vitro.

GML in olive (FIG. 1), palm (FIG. 2), or corn (FIG. 3) oil waspre-warmed to 37° C. to melt the GML and was then added to 1 mL of ToddHewitt (Difeo, Detroit Mich.) broth in round bottom tubes atconcentrations ranging from 10 μg/mL to 5000 μg/mL. The followingmicrobes were added to the tubes at the indicated concentrations:Staphylococcus aureus MNPE (methicillin sensitive, I×107/mL);Staphylococcus aureus MW2 (methicillin resistant, I×107/mL);Streptococcus agalactiae (I×107/mL); Gardnerella vaginalis (I×107/mL);or Candida albicans (I×106.)

The tubes were shaken at 37° C. at 200 revolutions per minute (RPM) instandard air for 18 hours. Plate counts were performed to determinecolony-forming units/mL (CFU/mL).

In the presence of as little as 10 μg/mL GML, G. vaginalis CFU/mL werereduced in all three vegetable oils tested: growth of both G. vaginalisand S. agalactiae was completely or nearly completely inhibited atlevels of 20 μg/mL GML or higher in all 3 vegetable oils tested.Additionally, the growth of C. albicans and both strains of S. aureuswas inhibited at 100 μg/mL GML and growth was completely inhibited byGML at 500 μg/mL and 5000 μg/mL GML, in all 3 vegetable oils tested.

Thus, GML was effectively anti-microbial when mixed with olive, palm, orcorn oil.

Example 2 Effect of GML on Microorganisms Growing as Biofilms

In order to assess the effect of GML on biofilms, S. aureus biofilmswere grown. S. aureus 128, an organism that expresses toxic shocksyndrome toxin-I (TSST-1), was inoculated at 107/0.1 mL onto the insideof pre-wetted dialysis tubing that had been tied off on one end. Atampon was then inserted into the dialysis tubing and the tubing wasimmersed under Todd Hewitt broth (Difeo Laboratories, Detroit, Mich.)containing 0.8% agar. The open end of the dialysis tubing remained abovethe agar surface such that the only source of nutrients for the growingmicrobes was media absorbed across the dialysis tubing. FIG. 4 showsbiofilm growth on tampon fibers and cellulose acetate dialysis tubingafter an 18 hour incubation, at 6000× (A) and 9000× (B) magnifications.

Tampon sacs were incubated in the solidified agar. At 4, 8, 12, and 16hours, tampon sacs were removed from the agar, sliced open, and weighedto determine fluid gain. TSST-1 was eluted by addition ofphosphate-buffered saline (PBS). The accumulated amount of TSST-1 wasquantified by first concentrating the eluted fluids by addition of 4volumes of absolute ethanol, then resolubilizing in distilled water, andanalyzing by Western immunoblot. FIG. 5 shows the increasing amounts ofTSST-1 present in the tampons over time. TSST-1 was not detected in thepresence of 5% GML (data not shown).

To directly assess the effect of GML on the formation of biofilms, 96well plastic microtiter plates were inoculated with approximately 106/mLof one of three strains of S. aureus (MN8, a methicillin sensitivestrain; MNWH, a methicillin resistant strain; or MW2, a methicillinresistant strain), or with non-typable Haemophilus influenzae. Wellswere cultured stationary at 37° C. for 24 and 48 hours (FIGS. 6-8). As acontrol, in one set of three wells for each microbe, the wells wereagitated 3 times by pipetting up and down. The bactericidal activity ofGML was determined by measuring CFU/mL in supernatants. After removal ofsupernatants, wells were washed three times with PBS to remove unboundcells, and were then treated with crystal violet for 30 minutes. Wellswere again washed three times with PBS to remove unbound crystal violet.Finally, wells were treated with ethanol to solubilizebiofilm-associated crystal violet. Absorbances at 595 nm were determinedby an ELISA reader to measure biofilm formation.

FIGS. 6, 7 and 8 provide the results of the study. Growth of all threeS. aureus strains was completely inhibited by GML at 500 μg/mL at both24 and 48 hours, as measured by CFU/mL (FIG. 6; dashed line indicatesstarting inoculum size; *significant reduction in mean CFU/mL comparedto starting inoculum, p<0.001.). In contrast, at 10 fold lower GMLconcentrations than necessary to inhibit bacterial growth, biofilmformation was significantly inhibited as measured by reduced crystalviolet staining of retained biofilm material in wells of the microtiterplates (FIG. 7; #significant reduction in mean absorbance at 595 nmcompared to no GML wells, p<0.01).

Similarly, GML was bactericidal against non-typable H. influenzae in thecontext of a biofilm at concentrations of 50 μg/mL (FIG. 8, top; dashedline indicates starting inoculum size; *significant reduction in meanCFU/mL compared to starting inoculum, p<0.001). In addition, GMLinhibited H. influenzae biofilm formation at concentrations as low as1.0 μg/mL, as measured at 24 and 48 hours (FIG. 8, bottom; #significantreduction in mean absorbance at 595 nm compared to no GML control,p<0.01).

In order to assess the effect of GML on previously formed biofilms,side-by-side wells that were not treated with GML throughout theincubation and that had high absorbances at 595 nm at 48 hours,indicating that a biofilm had formed in the well, were treated with 500μg/mL GML for 60 minutes at 37° C. Supernatants were removed andbactericidal activity was determined by measuring CFU/mL (FIG. 9, left;#significant reduction in mean absorbance at 595 nm compared to no GMLcontrol, p<0.01). Wells were then washed and stained with crystal violetas described above.

FIG. 9 provides the results of the study. After 48 hours, 500 μg/mL GMLkilled both S. aureus and H. influenzae (FIG. 9, left). Similarly, 500μg/mL GML nearly completely removed biofilm material attached to thewells as demonstrated by a loss of crystal violet staining inGML-treated wells (FIG. 9, right).

Example 3 Synergistic Effects of GML and EDTA on the Growth of E. coli

Previous studies indicated that Enterobacteriaceae species such as E.coli, as well as Pseudomonas aeruginosa, were resistant to GML'santibacterial effects, and suggested that the dense layer of LPS wasprotective from GML in these organisms. An additional study wasundertaken to determine if the addition of EDTA to a GML compositionwould enhance the antimicrobial properties of the GML composition.

E. coli (Watson strain) was adjusted to I.2×107/mL in Todd Hewitt media.Various concentrations of EDTA ranging from 0.05 M to 0.00005 M, in thepresence or absence of 100 μg/mL GML, were added to the wells. Cultureswere incubated with shaking (200 RPM) for 24 hours, at which timesamples were removed for plate counting.

The results of the study are shown in FIG. 10. EDTA alone inhibited thegrowth of E. coli somewhat, in a dose-dependent manner. The combinationof 100 μg/mL GML with EDTA showed increased anti-bacterial activity(*p<0.001; dashed line indicates starting inoculum size).

To further assess the combined effects of GML and EDTA, the experimentwas carried out as above, and the relative effects on the growth of E.coli of EDTA alone or the combination of GML and EDTA were assessed indirect comparison to GML alone. The results are provided in FIG. 11. Asin the experiment described above, EDTA alone inhibited the growth of E.coli to some extent at higher concentrations, while the combination of100 μg/mL GML with EDTA showed increased anti-bacterial activity, in adose-dependent fashion. GML alone did not exhibit any bactericidalactivity against E. coli. Therefore, GML and EDTA exerted a synergisticanti-bacterial effect.

Example 4 The Effect of pH on GML Activity Against PathogenicMicroorganisms

Because the presence of EDTA made E. coli susceptible to GML, it washypothesized that protonating the surface of E. coli or P. aeruginosamay increase GML activity through repelling divalent cations, therebydisrupting LPS integrity.

An experiment was conducted to determine the effect of GML in thepresence of various pH levels on the growth of E. coli. E. coli (Watsonstrain) was grown in Todd Hewitt media and adjusted to 107 CFU/mL. Usingacetate buffer, the pH of the cultures was adjusted to 5.0, 6.0, or 7.0.GML was added to cultures at concentrations of 5000, 50, or 0.1 μg/mL,and cultures were incubated at 37° C. with shaking (200 RPM). Sampleswere removed for enumeration of CFU/mL at 24 hours.

The results of the study are provided in FIG. 12. E. coli was notsusceptible to GML at pH 7.0, even when as much as 5000 μg/mL GML wasadded to the culture. However, E. coli was highly susceptible to GML ata pH of 6.0, as 50 μg/mL GML was bactericidal, and was even moresusceptible to GML at pH 5.0, as only 0.1 μg/mL GML was bactericidal inthese cultures. With each unit drop in pH, E. coli appeared to become500 times more susceptible to GML. (*p<0.001; dashed line indicatesstarting inoculum size).

A similar experiment was carried out to assess the effect of GML on thegrowth of Haemophilus influenzae at various pHs. 1 μg/mL GML had noeffect on the growth of H. influenzae at a pH of 7.0 (FIG. 13). However,at a pH of 6.0, 1 μg/mL GML completely abrogated the growth of H.influenzae at 4, 8, and 24 hours (FIG. 13).

The effect of GML at a range of concentrations and in the presence of arange of pH levels on the growth of Pseudomonas aeruginosa was alsodetermined. P. aeruginosa (strain PAO 1) was inoculated in Todd Hewittbroth at 5.7×106/mL. GML was added to cultures at a range ofconcentrations from 10 μg/mL to 5000 μg/mL, and the pH was adjusted to5.0, 6.0, or 7.0. CFU/mL was determined after 24 hours of incubation.The results of the study are shown in FIG. 14. At a pH of 6.0 or 7.0, noconcentration of GML was inhibitory for the growth of P. aeruginosa. ApH of 5.0 in the absence of GML was somewhat inhibitory for the growthof P. aeruginosa. However, the addition of GML to the cultures at a pHof 5.0 further inhibited P. aeruginosa growth in a dose-dependent manner(*p<0.001; dashed line indicates starting inoculum size).

The results of the study indicated that GML and a lowered pHsynergistically inhibited the growth of pathogenic microorganims.

Example 5 Effect of Non-Aqueous Gel on the Antimicrobial Activity of GML

Non-aqueous gels comprised of propylene glycol (73.55% w/w),polyethylene glycol 400 (25% w/w), and hydroxypropyl cellulose(Gallipot, St Paul, Minn.; 1.25% w/w), with or without GML, were heatedto 65° C. After solubliziation of components, the gels were diluted withTodd Hewitt broth to 10%, or 25%. GML alone was also diluted comparablywith Todd Hewitt broth to serve as an additional control. S. aureus(strain MN8, a toxic shock syndrome strain) was incubated at 37° C. withshaking (200 RPM) in the various concentrations of non-aqueous gel inthe presence of GML at concentrations ranging from 1 μg/mL to 5000μg/mL, or in the presence of GML alone at the same concentrations. After24 hours, plate counts (CFU/mL) were determined.

FIG. 15 shows the results of the study. GML inhibited the growth of S.aureus at a concentration of 100 μg/mL or above. The 10% and 25%,non-aqueous (NA) gel concentrations, as well as the range of GMLconcentrations, exhibited dose-dependent effects on the growth of S.aureus. GML in 25% non-aqueous gel had approximately 500-fold greateractivity than GML alone, and GML in 10% non-aqueous delivery vehicle hadapproximately 10-fold greater activity than GML alone (FIG. 15;*p<0.001; dashed line indicates starting inoculum size). Therefore, thecombination of the non-aqueous gel and GML had a potent antimicrobialeffect.

Example 6 Solubility of Tenofovir in GML Gels

Non-aqueous gels comprised of propylene glycol (73.55% w/w),polyethylene glycol 400 (25% w/w), and hydroxypropyl cellulose (1.25%w/w) at a range of pH from 4.0 to 4.5 were prepared. The anti-HIV drugTenofovir was added to the gels at a concentration of 10 mg/mL todetermine if the drug was soluble in the compositions.

The results of the study are shown in FIG. 16. Tenofovir (10 mg/mL) wasnot soluble in the non-aqueous gel at a pH of 4.0-4.3, but was solublein the non-aqueous gel at a pH of 4.4 or 4.5.

Example 7 Effectiveness of Various Forms of GML

Multiple forms of GML exist, including R or S optical isomers and GMLwith lauric acid ester linked in the 1/3-position or the 2-position ofglycerol. In order to test for potential differences between the opticalisomers, the R form of GML, which is commercially available, wascompared to a GML racemic mixture of the R and S forms. In order to testfor potential differences between GML with lauric acid linked todifferent glycerol positions, the commercially available purified2-position lauric acid GML was compared to a mixture of 2-position and1/3-position lauric acid GML. Antibacterial effects of these forms ofGML was assessed on S. pyogenes (strain 594).

The results of the study are shown in FIG. 17. The R form GML and themixture of R and S form GML had the same antibacterial activity (leftpanel; dashed line indicates starting inoculum size). However, the GMLform with lauric acid in the 2-position was 2-fold more active than themixture of GML forms (right panel; *p<0.001; dashed line indicatesstarting inoculum size). Therefore, the results indicated that GMLactivity depended not on chirality, but did to some extent depend on theposition of the lauric acid.

Example 8 Antibacterial Activity of GML Versus Lauric Acid

Bactericidal activity, as well as the ability to inhibit exotoxinproduction, of GML compared to lauric acid (a major cleavage product ofGML) was determined. S. aureus, an organism that produces glycerol esterhydrolase (GEH), and S. pyogenes, which does not produce GEH, weretested.

The results of the study with regard to the bactericidal activity of GMLand lauric acid are shown in FIGS. 18 and 19. GML and lauric acid wereincubated at the indicated concentrations with approximately 5×106CFU/mL S. aureus MN8 (FIG. 18) or S. pyogenes (FIG. 19) for 24 hours at37° C., in triplicate. For S. aureus, plates were incubated with shaking(200 RPM). For S. pyogenes, plates were incubated stationary in thepresence of 7% CO2. Plate counts were used to determine CFU/mL.Bactericidal activity was defined as the minimum concentration of GML orlauric acid required to reduce CPUs by at least 3 logs. GML wasbactericidal for S. aureus at 200-fold lower concentrations than lauricacid (FIG. 18; *p<0.001; dashed line indicates starting inoculum size).GML was bactericidal for S. pyogenes at 500-fold lower concentrationsthan lauric acid (FIG. 19; *p<O.001; dashed line indicates startinginoculum size). In addition, in comparing the bactericidal activity ofGML against the two organisms, GML was 5-fold more bactericidal for S.pyogenes than for S. aureus.

To determine the relative capacity of GML and lauric acid to inhibitsuperantigen production, S. aureus MN8 and S. pyogenes were cultured for8 hours in the presence of GML or lauric acid. TSST-1 production by S.aureus MN8 and streptococcal pyrogenic exotoxin A (SPE A) production byS. pyogenes were quantified by Western immunoblot analysis.

The results of the study with regard to superantigen production areshown in FIG. 20. Both GML and lauric acid significantly inhibitedexotoxin production by S. aureus MN8 and S. pyogenes at concentrationsthat were not growth inhibitory. However, the concentration of GMLrequired for inhibition of exotoxin production was lower for bothorganisms, compared to the concentration of lauric acid required toinhibit exotoxin production. GML inhibited production of S. aureusTSST-1 at a concentration of 0.2 μg/mL, and inhibited production of S.pyogenes SPE A at a concentration of 0.025 μg/mL, while lauric acidinhibited production of both TSST-1 and SPE A at a concentration of 2.5μg/mL. (*p<0.01).

To further assess the differences between S. aureus and S. pyogenes withregard to GML activity, GML was pre-treated by incubating overnight at aconcentration of 1000 μg/0.4 mL Todd Hewitt broth with 0.1 mL ofstationary phase sterile culture fluid from S. aureus MN8 or S.pyogenes.

The results of the study are shown in FIG. 21. Pre-incubation with S.aureus eliminated the antibacterial activity of GML against S. aureusMN8 as measured in the 24 hour assay described above. However,pre-incubation with S. pyogenes did not affect GML's antibacterialactivity. (*p<0.001; dashed line indicates starting inoculum size.)These results suggested that an esterase produced by S. aureus, such asGEH, inhibited GML activity.

Example 9 Development of Resistance to GML in S. Aureus

To determine if S. aureus developed resistance to GML, S. aureuscultures were treated with sub-optimal concentrations of GML (50 μg/mL)for one year. Each week, S. aureus strain MN8 was transferred to ToddHewitt agar plates containing 50 μg/mL GML and cultured for 48 hours.This sub-optimal GML concentration allows S. aureus to grow for 48hours. Organisms that grew were passed weekly onto new plates containing50 μg/mL GML, or were transferred to plates containing 100 μg/mL GML, aconcentration at which S. aureus cannot normally grow, for 24 hours. Inaddition, 50 g/mL GML plates were placed at 4° C. weekly to allow GML tocrystalize. Plates were analyzed for non-crystalizing zones aroundindividual S. aureus colonies, which is indicative of GEH cleavage ofGML.

Despite the fact that S. aureus exhibits rapid development of resistanceto many antibiotics, no S. aureus developed that was able to grow on 100μg/mL GML plates. Therefore, S. aureus did not develop resistance to GMLover the period of one year. In addition, no mutants that hadupregulated GEH production during the year of passage were identified(data not shown).

Example 10 Decolonization Studies

A study was undertaken to assess the ability of GML (5% w/v), formulatedin a non-aqueous gel, to decolonize the respiratory tract in humans, andto decolonize contaminated surgical incision sites in experimentalrabbits.

Three human subjects underwent swabs of the anterior nares in order toassess whether GML was capable of decolonizing the respiratory tract.Swabs were dipped in phosphate-buffered saline (PBS), which haspreviously been shown to result in the uptake of 0.1 ml of PBS, and thenused to swab anterior nares of each subject. The swabs were rotatedaround each nare up to the nasal bone 3 times. Colony-forming units ofmicrobes from swabs were determined by plate counts on blood agar andmannitol salt agar. The anterior nares were then treated in the same waywith swabs that had been dipped one time in GML gel. The anterior naresof each participant were swabbed at designated time periods for up to 24hours, and swabs were cultured for S. aureus and coagulase-negativestaphylococci.

The data obtained from this study are shown in FIG. 22. Data were logtransformed prior to performing statistics due to the high variabilityin CPUs present in the three subjects. The right and left anterior naresof the three subjects contained an average log CFU/mL S. aureus of 1.6or 1.5, respectively, prior to GML treatment. GML treatmentsignificantly (p<0.05 by Student's t test analysis) reduced S. aureuscounts in both nares to 0 CFU/mL at all tested time-points aftertreatment, including the 24 hour time-point. The right and left anteriornares of the three subjects contained an average log CFU/mL ofcoagulase-negative staphylococci of 3.9 and 3.8, respectively, prior toGML treatment. GML treatment significantly (p<0.05) reducedcoagulase-negative staphylococcal counts in both nares at testedtime-points of 4 hours or more after treatment, including the 24 hourtime-point.

For one subject, the persistence of reduced CPUs of both S. aureus andcoagulase-negative staphylococci were tested over 3 days. S. aureuscounts remained at 0 for the entire 3 day test period. The CPUs ofcoagulase-negative staphylococci also remained low for the entire timeperiod (initially there were 560 and 880 CFU/mL in the right and leftnares, respectively; after 3 days, 8 and 0 CFU/mL of coagulase-negativestaphylococci were detected in the right and left nares, respectively;data not shown).

Studies were next performed to assess whether 5% GML gel coulddecolonize teeth of oral aerobic bacteria. Human volunteers were swabbedwith a PBS-saturated swab across the teeth and gum lines on the leftside of the mouth and tested for CFU/mL of total bacteria subsequentlygrown on blood agar plates. The same individuals were then swabbed withGML gel by swabbing the gel across the teeth and gum lines on the rightside of the mouth. Enough gel was used to coat the entire surface areaof the teeth and gum lines. Thirty minutes after treatment, theparticipants were swabbed with PBS-saturated swabs on the right side andtotal CFU/mL were determined.

To ensure that the data obtained did not differ simply due to removal ofbacteria by the initial swab, different sides of the teeth and gum linesfor the pre- and post-treatment swabs were used. Bacterial counts onboth sides of the teeth and gum lines were presumed to be approximatelyequal, and a pre-test swab confirmed that this was the case.

The results of the study are shown in FIG. 23. Data were log transformedto account for high variability in CFU/mL among subjects. There was a >5log reduction in CPUs between the pretreatment swabs and thepost-treatment swabs, indicating the GML killed the bacteria adhering tothe teeth, which were primarily oral streptococci. Since GML waseffective in reducing counts, the data also suggested that GML gel waseffective in removing and/or killing bacteria in biofilms, which wouldbe expected to be present on teeth. The data were highly significantlydifferent as tested by Student's t test analysis (p<0.001).

In a final study, S. aureus strain MN8 (I×1010 CFU) was used to coatsurgical incision sites of three rabbits per group. The surgicalincision sites were 4 cm subcutaneous incisions —that had been closedwith 4 silk sutures (Ethicon, Cornelia, Ga.). After closing, GML 5% w/vnon-aqueous gel was swabbed onto the incision sites of three animals,and PBS was swabbed onto the incision sites of control animals. EnoughGML gel was swabbed to provide a uniform coating of the surface. After24 hours, the rabbits were examined for inflammation (as determined byredness in the incision sites) and total CFU/mL that could be obtainedby swabbing the incision sites with PBS-saturated swabs.

The results of the study are shown in FIGS. 24 and 25. Rabbits that wereswabbed with PBS had an average of 8.8 log CFU/mL of S. aureus at the 24hour time-point (FIG. 24) and obvious inflammation at the surgical site(FIG. 25). In contrast, in rabbits that had been treated with GML, noCPUs were detectable at the 24 hour time-point (FIG. 24; p<<0.001 byStudent's t test analysis of log transformed data) and less inflammationat the surgical site (FIG. 25).

Collectively, the data presented in this study showed that a 5% GMLnon-aqueous gel could be used effectively to reduce colonization of thenasal and oral cavities of humans and rabbit surgical incision sites bypotential pathogens.

Example 11 Clinical Efficacy of GML in Vaginal Infection

The following prophetic example provides a proposed clinical studywherein the relative effectiveness of a composition comprising GML, avegetable oil, and a pharmaceutically acceptable topical carrier will bedetermined for the treatment of BV or VVC. This prophetic example isintended to illustrate the principles of the present invention.

The study design is a single-center, double-blind study of 60 subjects,20 subjects per arm. Subjects will be stratified based on type ofvaginal infection (BV, VVC, or both) and age.

Study subjects will be females aged 18-50 with BV or VVC (as determinedby gynecological exam conducted during the screening visit) who havesigned Informed Consent. Pregnant women, menstruating women, and womenwho have had a systemic infection or have used a vaginal anti-microbial,anti-inflammatory, or immunosuppressant medication within the previous 4weeks will be excluded.

Study endpoints and treatment evaluation: A vaginal swab will becollected at the baseline visit. Subjects will vaginally self-administerone of the following every 12 hours for two days, for a total of 4doses: 0% (vehicle control), 0.5%, or 5% GML in olive oil. Vaginal swabswill be collected 12 and 48 hours after the final dose of study drug orvehicle control. Colony Forming Units (CFU) will be determined forLactobacillus, G. vaginalis, and Candida on baseline and treatmentfollow-up vaginal swabs. Subjects will be followed for a period of 3months and all adverse events including clinical findings andopportunistic infections will be recorded.

The results of the proposed study will be employed in the development ofa clinical protocol for the treatment of BV or VVC.

Example 12 Clinical Use of GML in Urinary Tract Infection

The following prophetic example is intended to illustrate circumstanceswherein the formulations herein disclosed are indicated.

A subject at risk of urinary tract infections (e.g., for example, awoman or an elderly individual) may apply a composition comprising 50μg/mL GML to a sponge, and then apply the sponge to the area of theexternal urethral opening or orifice. The composition may be appliedonce per day to prevent urinary tract infections. The composition mayadditionally comprise an accelerant such as EDTA and/or a non-aqueousgel.

Example 13 Clinical Use of GML in Cellulitis

The following prophetic example is intended to illustrate circumstanceswherein the formulations herein disclosed are indicated.

A subject that has been diagnosed with cellulitis may topicallyself-administer a composition comprising 5 pig/mL GML and 25%non-aqueous gel in a pharmaceutically acceptable topical carrier to thesite of the skin infection, twice per day until the infection resolves.If medically indicated, the patient may also be administered anantibacterial agent.

All, documents, patents, patent applications, publications, productdescriptions, and protocols which are cited throughout this applicationare incorporated herein by reference in their entireties for allpurposes.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

1. A method of treating or preventing a microbial or viral skininfection, a microbial or viral infection of a wound, or a microbial orviral infection of surgical incision cite in a subject in need thereof,the method comprising the step of topically administering an effectiveamount of a non-aqueous composition comprising i) glycerol monolaurate(GML) and a vegetable oil, or ii) GML and a non-aqueous gel to treat orprevent the microbial or viral skin infection, the microbial or viralinfection of a wound, or the microbial or viral infection of surgicalincision cite in the subject.
 2. The method of 1, wherein thenon-aqueous composition further comprises a pharmaceutically acceptabletopical carrier.
 3. The method of 1, wherein the non-aqueous compositionfurther comprises an accelerant selected from an organic acid, achelator, an antibiotic agent, an anti-fungal agent, an anti-viralagent, or a combination thereof.
 4. The method of 1, wherein thevegetable oil is palm oil, olive oil, corn oil, canola oil, coconut oil,soybean oil, wheat germ oil, or a combination thereof.
 5. The method of1, wherein the non-aqueous gel is comprised of propylene glycol,polyethylene glycol, cellulose derivative, or a combination thereof. 6.The method of 1, wherein GML comprises about 0.001% (w/v) to about 10%(w/v) of the composition.
 7. The method of 1, wherein GML is present ata concentration of about 10 μg/mL to about 100 mg/mL.
 8. The method of1, wherein the pH of the non-aqueous composition is from about 4.0 toabout 4.5.
 9. The method of 1, further comprising administering a secondactive agent selected from an antibiotic, an anti-fungal agent, or ananti-viral agent.
 10. The method of 1, the subject has a bacterialinfection.
 11. The method of claim 10, wherein the bacterial infectionis a Staphylococcus infection.
 12. The method of claim 10 wherein thebacterial infection is methicillin-resistant Staphylococcus aureus. 13.The method of 1, wherein the bacterial infection is a Streptococcusinfection.
 14. The method of 1, wherein the subject has a viralinfection.
 15. The method of 14, wherein the viral infection is humanimmunodeficiency virus.
 16. The method of 14, wherein the viralinfection is herpes simplex virus.
 17. The method of 1, wherein thesubject has a fungal infection.
 18. The method of 1, wherein the subjectis a human.
 19. The method of 1, the method further comprising using asponge, wipe, or swab to topically apply the non-aqueous composition.